This invention relates to a method for preparing a therapeutic agent that upon ingestion decreases intestinal cholesterol absorption in man and specifically inhibits or decreases intestinal cholesterol absorption by inhibiting the pancreatic cholesterol esterase catalyzed hydrolysis of naturally occurring and ingested cholesterol esters and by inhibiting the cholesterol esterase facilitated uptake of free cholesterol.
The invention is based upon the discovery that pancreatic cholesterol esterase is an important contributor to overall dietary cholesterol absorption because (1) cholesterol derived from cholesterol esters is preferentially absorbed compared to free cholesterol; (2) cholesterol esterase enhances the absorption of free cholesterol and (3) dietary cholesterol and/or cholesterol esters induce the mRNA and level of enzymatic activity of cholesterol esterase in the pancreas in a newly discovered intestinal-pancreatic cycle for the absorption of cholesterol. U.S. Pat. Nos. 5,173,408 and 5,063,210 describe the importance of cholesterol esterase in the dietary uptake of cholesterol and also disclose methods for inhibiting cholesterol esterase. Thus, the surprising usefulness of inhibiting cholesterol esterase has demonstrated a new need for potent (Ki less than 5 .mu.M) and safe inhibitors of cholesterol esterase.
Many physical ailments are attributed at least in part to high levels of serum cholesterol. Atherosclerosis, for example, is a leading cause of death in the United States and high serum cholesterol concentrations are associated with increased risks of fatal atherosclerotic events. The discovery that the cholesterol esterase enzyme plays a role in intestinal cholesterol absorption has led to attempts to attenuate intestinal cholesterol absorption in man by inhibiting the action of the cholesterol esterase enzyme. As a result of these findings, there is now an important need to develop human pancreatic cholesterol esterase inhibitors, especially those that are not absorbed and are essentially nondegradable. The pharmacology of various polysaccharides has been investigated. Cook and Cammarata, 1963, Arch. Int. Pharmacodyn. 144:1. In particular, crude sulfated amylopectin has been taught in U.S. Pat. No. 4,150,110 as an anti-ulcer agent, but its property as a cholesterol esterase inhibitor has not been recognized.
Sulfated dextran of low molecular weight has been recognized for use in the treatment of hyperlipemia and as an orally administered anticoagulant. British Patent No. 953,626. In Japan, low molecular weight sulfated dextran (MDS) at a dose of 1800 mg/day has been used to reduce serum cholesterol levels by activating a blood enzyme lipoprotein lipase. Goro et al, 1987, J. Clin. Biochem. Nutr. 2:55-70. As demonstrated by carbon-14 labelling studies, the low molecular weight of this bacterial dextran, (7-8,000 Daltons), allows the sulfated dextran to be absorbed by the intestine Drugs In Japan (Ethical Drugs, 10th ed. 1986). MDS was developed for this property of intestinal absorption as indicated by the claim that a faster reduction in serum lipids can be obtained by intravenous administration of this agent with clearance of serum lipemia due to activation of plasma lipoprotein lipase. Clearly this route of administration will not lead to effects on inhibiting cholesterol esterase in the intestine. Absorption of MDS can lead to a variety of side effects, most notably, anticoagulant effects that must be monitored. This preparation has not been known to inhibit cholesterol esterase and it is sulfated randomly and at various ring positions. High molecular weight dextran sulfate has been excluded from development by others because of its lack of absorption and its attendant inability to activate serum lipoprotein lipase.
More recently, it has been discovered that crude non-absorbable polysaccharides sulfated at the three position of the glucopyranose ring are effective as inhibitors of cholesterol esterase. See U.S. patent application Ser. No. 08/121,369. Useful 3-sulfated polysaccharides may be derived from the synthetic sulfation of polysaccharides from various natural sources including seaweeds.
Methods for preparing sulfated polysaccharides are also known in the art. For example, U.S. Pat. No. 3,624,069 describes the sulfation of cellulose with a sulfur trioxide/lower n-dialkyl amide sulfation complex. U.S. Pat. No. 4,480,091 describes a process for preparing cellulose sulfate esters in a three step process. Finally, U.S. Pat. No. 4,814,437 describes a method for preparing sulfated polysaccharides by subjecting the polysaccharide to a reducing step prior to sulfation.